Project Summary/Abstract Though curable, malaria is a persistent global health crisis, with over 200 million debilitating cases a year and half a million deaths, mostly in children under five. Plasmodium falciparum (Pf) has developed resistance to all antimalarials, including the mainstay artemisinins (ARTs). ART and its semi-synthetic analogs are considered essential for malaria treatment. ART resistance is widespread in Southeast Asia and there are increasing reports of ART resistance in Africa. Combination therapy is a backbone for treatment of tuberculosis, cancer, HIV and malaria. Despite the huge success of the combination therapy, its efficaciousness can be derailed as a two-drug combination can become de facto monotherapy under certain unavoidable situations. Moreover, extended exposure of Pf to ART induces multidrug tolerance. We recently showed that inhibitors specific for Pf proteasome (Pf20S) can kill Pf in each stage of its life cycle and synergize with ART, overcoming ART resistance. In this proposal, we hypothesize that what we call an artezomib (ATZ) ? a covalent hybrid of an ART analogue and a Pf20S inhibitor ? can enhance ART action and minimize the emergence of resistance to both components. We predict that Pf will active the ART component of ATZ, which, like ART itself, will bind to Pf proteins; Pf20S will generate ATZ-modified oligopeptides; and the peptides' extended contact with the Pf20S active site will augment the binding of the Pf20S inhibitor component of ATZ and overcome the decreased binding that might result from Pf20S point mutations. Consistent with this, we have synthesized ATZs that are more potent Pf20S inhibitors than their component Pf20S inhibitor, and these ATZs are potent against wild type, Pf20S inhibitor-resistant and ART-resistant K13 Pf. We now aim to explore the mechanism of action of AZTs and develop more drug-like AZTs with a long residence time on target so as to kill parasites when they exit the stage of their life cycle in which they are intrinsically resistant to ARTs. .